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SRX1012871: GSM1669393: rosettes_wildtype_watered_H3K4me3_rep3; Arabidopsis thaliana; ChIP-Seq
1 ILLUMINA (Illumina Genome Analyzer II) run: 20.4M spots, 715.7M bases, 350.9Mb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana [ChIP-Seq]
show Abstracthide Abstract
Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained largely transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which even under normal conditions exhibited lower H3T3ph levels in pericentromeric regions, and a few transposons and repeat sequences showed modest transcriptional activation. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5’ genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s). Overall design: Columbia-0 and double mutant at3g03940/at518190 knockdown plants were grown in 12 hr light for 3 weeks in pots with soil covered with miracloth to prevent soil contamination of leaf tissues. Control was kept in normal watered state, for other samples (peg) drought stress was induced by treatment with 30% Polyethylene glycol (PEG 6,000) for 5 hours. Pulldowns on H3, H3K4, and H3T3 were performed on all samples with 3-4 replicates.
Sample: rosettes_wildtype_watered_H3K4me3_rep3
SAMN03571032 • SRS924691 • All experiments • All runs
Instrument: Illumina Genome Analyzer II
Strategy: ChIP-Seq
Selection: ChIP
Layout: SINGLE
Construction protocol: Watered or water deficit treated vegetative tissues were fixed in 1% formaldehyde for 10 min after vacuum infiltration of the tissues in phosphate buffered saline (PBS) with 0.007% silwet. Fixation was stopped by quenching with 125 mM glycine. Next, the tissue was rinsed twice in ice cold water, blotted dry and flash frozen in liquid nitrogen. Chromatin was isolated and resuspended in 300 ml nuclei lysis buffer and sonicated for four 15 sec pulses using a Branson sonifier 450 at an output between 10 – 15%. 100 ml of sheared chromatin was diluted in 900 ml ChIP dilution buffer and precleared, using 5 ml of protein A Dynabeads (Invitrogen, Carlsbad, CA). Protein A beads were removed on a magnet, and the precleared chromatin was incubated overnight at 4 °C with antibodies against either mono-methylated H3K4 (10 mL, Abcam ab8895), dimethylated H3K4 (6 mL, Upstate 07-212), or trimethylated H3K4 (6 mL, Abcam ab8580). Next the antibody/chromatin complex was precipitated by incubating the mix with 40 ml protein A beads for 1 hour followed by collection of the beads on a magnet. The beads were washed and chromatin eluted and de-crosslinked using procedures. DNA was recovered by phenol/chloroform extraction and ethanol precipitation in the presence of Novagen pellet paint (CN Bioscience) and resuspended in 30 mL of TE buffer. Purified DNA was end modified, ligated to amplification primers, amplified, and sequenced according to the manufacturer’s protocols (Illumina, San Diego, CA).
Experiment attributes:
GEO Accession: GSM1669393
External link:
Runs: 1 run, 20.4M spots, 715.7M bases, 350.9Mb
Run# of Spots# of BasesSizePublished


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